The ability to suppress gene expression with RNA homologous to targeted gene sequences has greatly increased demand for large scale production of such RNA. However, the chemical fragility of RNA limits commercial development of many of these techniques. Large scale production of purified RNA is constrained by the high costs associated with in vitro synthesis methods and by the low yields and complex processing requirements associated with in vivo methods.
The susceptibility of RNA to enzymatic and environmental degradation varies widely depending on the nature of the RNA molecule. Single-stranded RNA (ssRNA) is extremely sensitive to degradation and in vivo production of such molecules requires use of production strains lacking endogenous RNAses and benefits by coupling production of the RNA to encapsidation within viral capsid shells to produce Virus-Like Particles (VLPs). Encapsidation reduces degradation of RNA during production and allows more aggressive treatment during purification. VLPs effectively preserve such fragile RNA from degradation by sequestering the RNA within a relatively inert protein shell. Double stranded RNA (dsRNA) are somewhat less susceptible to degradation by cellular and environmental RNAses, although the highest in vivo yields of dsRNA also involve production strains lacking RNAses and many dsRNA also benefit from encapsidation. Unfortunately, the semi-rigid nature of the double-stranded stem region of dsRNA limits the range of dsRNA that can be encapsidated since the length of the double-stranded stem structure cannot exceed the interior diameter of the capsid.
In the course of exploring techniques for increasing the range of dsRNA stems that can be encapsidated, the inventors discovered that under certain conditions a large amount of unencapsidated dsRNA can be recovered directly from cell lysates, but only when the host cells co-express capsid protein or specific portions thereof. The presence of high quantities of intact unencapsidated dsRNA in crude cell lysates represents a significant advance in the ability to generate commercial quantities of such RNA for gene suppression and other activities.
Dimers of bacteriophage capsid proteins such as those of the leviviruses MS2 or Qβ recognize and bind with affinity to cognate pac sequences. Such pac sequences comprise approximately 19-21 nucleotides comprising an 8 base pair bulged stem and 4 base loop capable of forming a discrete hairpin structure. Such sequences may be referred to herein as pac-sites, pac sequences, pac-site sequences, pac-site hairpins, or pac-site hairpin sequences. The interaction of capsid dimers with their cognate pac site hairpin is well-characterized and is known to play at least two key roles in the bacteriophage life cycle. Binding of capsid dimers to the cognate pac sites is required for programmed translational repression of the phage encoded replicase, which is only expressed early in infection. In addition, capsid protein binding to both to pac-site sequences and multiple dispersed and degenerate RNA sites with cognate coat protein affinity (the packaging signals described by Dykeman et al., Packaging Signals in Two Single-Stranded RNA Viruses Imply a Conserved Assembly Mechanism and Geometery of the Packaged Genome J. Mol. Biol. 425:3235-3249 (2013)) are required for proper assembly into progeny bacteriophage.
The interaction of capsid dimers with cognate pac sites is the subject of a number of different published in vitro and in vivo methods designed to allow encapsidation of heterologous RNAs of various kinds by associating the desired cargo molecule with pac site sequences, either by direct covalent linkage or by various affinity methods. The present invention differs markedly from such approaches in that it comprises co-expression of capsid proteins to produce unencapsidated dsRNA rather than encapsidated RNA. Further, the present invention allows in vivo production of dsRNA entirely lacking pac or any recognized dispersed and degenerate RNA sites with cognate protein affinity. In vivo production of such dsRNA molecules is highly desirable since reducing extraneous sequence reduces the chance of off-target RNAi interactions.